Extracellular Optical Recording Configuration for Neuronal Action Potential Detection by using Surface Plasmon Resonance: Preliminary Experiment

Proceedings of the 2nd International IEEE EMBS Conference on Neural Engineering Arlington, Virginia · March 16 - 19, 2005

Extracellular Optical Recording Configuration for Neuronal Action Potential Detection by using Surface Plasmon Resonance: Preliminary Experiment Hyoungwon Baac1,3, Sang Beom Jun1,2, James N. Turner2, William Shain2, Karen L. Smith2, Michael L. Shuler3, and Sung June Kim1 1

Nano-Bioelectronics and Systems Research Center, Seoul National University, Seoul 151-742, Korea 2 Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509 USA 3 School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA

problems associated with both microelectrodes and fluorescence dyes while gaining the inherent advantages of these methods. Surface Plasmon Resonance (SPR) can be induced at the interface between metal and dielectric medium [10]. It is generated mostly by laser beam in forms of collective electron oscillation along the interface. The SPR condition depends on dielectric properties of both metal and dielectric medium. Such properties at the interface can be electrochemically modulated by external electric field application. Electrochemical SPR in Keywords─action potential recording, neuron, surface plasmon aqueous solution has been widely used to study the metalresonance electrolyte interface [11-12]. Recently, cyclic voltammetry showed that the change in charge density less than hundred I. INTRODUCTION µC/cm2 can be detected by resonance angle shift on the order of ∆θ ~10-3 where 0 to –0.3 or 0 to 0.3 voltage steps are applied Multi-channel simultaneous recording of neural action [12]. The variation of voltage and charge would be smaller potential is on fundamental demand for both understanding during action potential passage of neurons. However, highly neuron-neuron communication in network level and developing sensitive SPR configurations have been steadily developed neural engineering devices or sensors [1,2]: for example, neural enabling to measure ∆θ ~10-5 or more [13]. This offers a new probes for brain-computer interface and in vitro neural cell- opportunity for more sensitive applications. based biosensors. Especially for in vitro extracellular recording, It is challenging to apply SPR to measure such small common approaches to multi-channel configuration are to use neuronal activity as action potentials. Optical recording based either microelectrode arrays [3,4] or voltage-sensitive on SPR will allow direct detection of electrical signal at metalfluorescence dyes [5-7]. Microelectrode techniques have electrolyte interface, without use of fluorescence dyes. The difficulties associated with recording from microelectrode sites signal will not be interfered by other mechanisms during signal in terms of ‘electrode impedance’ [8]. The measured signal transmission since the recording spot is wirelessly addressed by quality is limited by electrical characteristics of electrode site laser beam. Multi-channel simultaneous recording may also be including charge delivery capacity and electrode area. Signal done simply in photodetector stage utilizing photodiode array transmission may be disturbed by various mechanisms such as or CCD apparatus. The imaging application of SPR has already biological and chemical artifacts as well as capacitive coupling been studied for applications to DNA [14], protein [15], and by power line. In contrast, the optical recording scheme using cell [16]. Here, as preliminary work, we investigate voltage-sensitive dyes is free from terms of impedance. Multi- electrochemical modulation of SPR effect without neurons. channel recording process is realized in a relatively simple way Issues in recording neural action potential and ways to because multiple signal acquisition is performed in overcome limitations are discussed with combining the SPR photodetector stage far from neuron-substrate interface [9]. optical configuration. Moreover, a site of choice for recording is not restricted by predetermined location. However, the change in membrane Ⅱ. EXPERIMENTAL potential should be mediated by electro-optical properties of voltage-sensitive organic dyes that limit the signal quality. The A. Electrochemical SPR Setup use of fluorescence dyes restricts recording duration due to fluorescence decay. Their molecular toxicity may potentially Fig. 1 illustrates a brief schematic of electrochemical SPR affect physiological environment. Therefore, it would be setup. Kretschmann configuration [10] with 632.8 nm He-Ne valuable to develop more efficient way to overcome the Abstract─We propose an extracellular optical recording configuration for neuronal action potential detection by using Surface Plasmon Resonance (SPR). The method does not use fluorescence dyes but still taking advantages of optical recording. As a preliminary experiment, the electrochemical SPR effect without neurons was investigated. This showed that a high resolution SPR setup (10-4~10-5 angular shift or more) is required to overcome noise and improve SPR signal. Several issues for neural signal recording are addressed to modify SPR optical configuration.

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(a) Fig. 1. Schematic view of SPR setup. Prism is mounted on motorized rotation stage. Voltage input is given at 1 kHz by function generator. Signal conditioning is performed through two stages (see text for detail).

laser beam is used to generate SP. High refractive index prism and glass slide (SF-10) are attached by index matching liquid. Titanium layer for adhesion is evaporated over the glass slide by 1 nm thickness, which is followed by 49 nm thick gold layer formation. Two gold-coated glass slides are assembled into a sandwich-type sample with 70 µm gap which is filled by Phosphate Buffer Solution (PBS) with pH=7.4. The prism is mounted on motorized rotation stage (Newport, SR50PP). Its angular rotation is digitally controlled. Electric function generator (Hewlett Packard, 3314A) is used to apply 1 kHz electric field across the sample. The reflected beam irradiates to silicon photodiode (Hamamatsu, S2387-1010R). The photodiode current output is converted to voltage and amplified by ×100,000 with 6 dB bandpass filter at 1 kHz cutoff frequency (low and high cutoff points are same) at the first stage (Stanford Research Systems, SR570), and again by ×1,000 with 20 dB bandpass filter at 0.3-1 kHz (Dagan, Model 2400). Finally, the signals are measured by analog oscilloscope (BK Precision, Model 2160). B. Electrochemical Modulation Condition Fig. 2(a) shows an angular profile of SPR in our setup. Differential reflectance in Fig. 2(b) shows a maximum variation point in reflectance, ~55.7 °(dotted line). Incidence angle of laser beam is fixed to 55.7 ° while we modulate the SPR by external electric field. Three different voltages, 50 mV, 30 mV, and 10 mV (peak to peak, rectangular waveform) are applied with the same frequency, 1 kHz. Ⅲ. RESULTS AND DISCUSSION

(b) Fig. 2. SPR curve: (a) Reflectance and (b) Differential reflectance. Differential reflectance is directly calculated from (a).

modulated reflectance (green) for three applied voltages: 50mV, 30 mV, and 10 mV. Light modulation was distinguishable for 50 mV and 30 mV cases although signal profiles were not fully consistent due to noise. Typically, more than 15 mV of voltage application allowed visual distinction in oscilloscope screen. Signal distortion was severe under 10 mV. We believe that the signal distinction should become clear at least near a few mV as we consider small action potential spikes observed in common extracellular recording process by microelectrode. The preliminary result in Fig. 3 reveals that the signal quality should be improved to overcome fundamental noises such as laser beam fluctuation, thermal noise in the photodiode, and mechanical vibration probably involved. B. Issues for better signal quality and neural action potential recording

A. Electrochemical Modulation of SPR Effect SPR signals were electrochemically modulated following the applied field frequency. Fig. 3 shows typical time traces of

The current SPR optical setup allows to measure the resonance angle shift of ~10-3. For detecting small signals

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single neuron, may disturb the efficient modulation of SPR signals because it involves the area without the neuron. IV. SUMMARY We propose an extracellular optical recording configuration for neuronal action potential detection by using SPR. The method does not need fluorescence dyes but still has the advantages of optical recording. As a preliminary experiment, the electrochemical SPR effect was investigated without neurons. This showed that high resolution SPR setup is required to overcome noises and improve SPR signal quality. We expect that an advanced SPR configuration using multi-cell photodiodes will be a promising solution capable of detecting 10-4~10-5 angular shift. Several issues for neural signal recording were also addressed to modify SPR optical configuration. ACNKOWLEDGMENT

Fig. 3. Electrochemical modulation of SPR reflectance. Green lines are light intensity (arbitrary scale) for three different applied voltages. White rectangular wave is overlapped for guiding the time trace of applied voltage (arbitrary scale). All signal traces are measured by oscilloscope and captured by digital camera (shutter speed = 1/1000 sec). Blurred images are due to the limitation of camera shutter speed.

This work was supported by Korea Science and Engineering Foundation (KOSEF) through Nano Bioelectronics and Systems Research Center (NBS-ERC) in Seoul National University, Nanobiotechnology Center (NBTC), an STC Program of the National Science Foundation (Agreement No. ECS-9876771), and the Cornell Nanoscale Facility (Agreement No. ECS-9731293). REFERENCES

electrochemically modulated, a highly sensitive and reliable setup is required, which can measure 10-4~10-5 angular shift. Such sensitivity will be the level endurable against most of fundamental noises. Recently, SPR configurations using multicell photodiodes have been proposed [11-13]. Briefly, such scheme is to acquire two or four signals from multiple elements of photodiodes and compensate each other to discard noises included in signals. This approach enabled to measure highly sensitive signals, ~10-5 angular shift or more. For neural signal recording, signal conditioning apparatus should be connected with each photodiodes: 4-pole Bessel filters and high gain transimpedance amplifiers for each photodiodes [9]. This means that two or four photodiodes are required for single spot measurement. Neuronal cell culture on gold substrates will make an adverse environment for SPR configuration in comparison to the case without neurons. Various biological species secreted from neurons will form layers over gold surface in nonuniform way. This may disturb to obtain consistent SPR signals because the SPR is sensitive to surface uniformity associated with light scattering and absorption. Sensing area is also an issue that should be considered for single neuron analysis. Electrochemical modulation by single neuron needs to be isolated on the sensing spot from other neurons. Incident laser beam can be focused by lens to less than 10 µm in diameter fitting into the size of neuronal cell body. The excessive dimension of focused beam, greater than

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